Calcium-dependent conformation of E. coli alpha-haemolysin. Implications for the mechanism of membrane insertion and lysis.
Previous studies from this laboratory had shown that calcium ions were essential for the membrane lytic activity of E. coli alpha-haemolysin (HlyA), while zinc ions did not sustain such a lytic activity. The present data indicate that calcium-binding does not lead to major changes in the secondary structure, judging from circular dichroism spectra. However binding to Ca2+ exposes new hydrophobic residues at the protein surface, as indicated by the increased binding of the fluorescent probe aniline naphtholsulphonate (ANS), and by the increased tendency of the Ca2+-bound protein to self-aggregate. In addition zinc ions are seen to decrease the thermal stability of HlyA which, according to intrinsic fluorescence and differential scanning calorimetry data, is stable below 95 degrees C when bound to calcium, while it undergoes irreversible denaturation above 60 degrees C in the zinc-bound form. Binding to phosphatidylcholine bilayers is quantitatively similar in the presence of both cations, but about one-third of the zinc-bound HlyA is released in the presence of 2 M NaCl. Differential scanning calorimetry of dimyristoylglycerophosphocholine large unilamellar vesicles reveals that Zn2+-HlyA interaction with the lipid bilayer has a strong polar component, while Ca2+-HlyA appears to interact mainly through hydrophobic forces. Experiments in which HIyA transfer is measured from phospholipid vesicles to red blood cells demonstrate that Ca2+ ions promote the irreversible binding of the toxin to bilayers. All these data can be interpreted in terms of a specific Ca2+ effect that increases the surface hydrophobicity of the protein, thus facilitating its irreversible bilayer insertion in the fashion of intrinsic membrane proteins.
Bakás
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L
Grupo Biomembranas (Unidad Asociada al C.S.I.C.), Departamento de Bioquímica, Universidad del País Vasco, Bilbao, Spain.
Veiga
M P
MP
Soloaga
A
A
Ostolaza
H
H
Goñi
F M
FM
D001426:Bacterial Proteins / Q000737:chemistry*; D002118:Calcium / Q000494:pharmacology*; D002413:Cations, Divalent; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins / Q000737:chemistry*; D006358:Hot Temperature; D008051:Lipid Bilayers / Q000737:chemistry*; D010446:Peptide Fragments / Q000032:analysis; D010713:Phosphatidylcholines; D011487:Protein Conformation; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D014357:Trypsin; D015032:Zinc
D001426:Bacterial Proteins; D002413:Cations, Divalent; D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins; C036507:Hlya protein, E coli; D008051:Lipid Bilayers; D010446:Peptide Fragments; D010713:Phosphatidylcholines; D014357:Trypsin; D015032:Zinc; D002118:Calcium